Fresh embryo transfer in the cleavage and blastocyst stages and pregnancy outcomes: A retrospective cross-sectional study

Abstract Background Advances in cell culture media have led to a trend of embryo transfer from the early cleavage stages to blastocyst stage. Objective The study aims to compare the effects of fresh embryo transfer in cleavage and blastocyst stage on pregnancy outcomes. Materials and Methods This cross-sectional study was conducted on 1422 cases referred to the Umm-al-Banin Infertility Clinic Center, Dezful, Iran, between July 2013 and December 2020 who were candidates for in vitro fertilization/intracytoplasmic sperm injection for fresh embryo transfer. A total of 1246 cases were divided into 4 categories on days 2-5 or 6. Chemical and clinical pregnancy, abortion, multifetal pregnancy, ongoing pregnancy, and live birth rates were analyzed. Results Fresh embryo transfer was performed in 28.5% of the cases on the 2 nd day, 45.8% on the 3 rd , 15.3% on the 4 th day, and 10.4% on days 5 or 6. The overall clinical pregnancy and live birth rates were estimated at 20.6% and 17.6% in the cleavage, and 17% and 14% in the blastocyst stages, respectively. However, no significant difference was observed in either group. In addition, there was no significant difference between groups in terms of the abortion, multifetal pregnancy, and ongoing pregnancy rate (p > 0.05). Conclusion According to the results, the consequences of pregnancy in fresh embryo transfer at the blastocyst stage were not superior to embryo transfer at different stages of the cleavage process.


Introduction
Infertility associated with reproductive disorders is a pervasive problem in human societies, defined as the absence of pregnancy after 1 yr of regular intercourse without using contraception (1). In recent decades, the increasing demand of infertile couples for fertility has urged the development of various fertility technologies in treating fertility in vitro.
Embryo transfer and subsequent in vitro fertilization (IVF) are classically done at the cleavage on day 2 or 3 after fertilization, which is in the cell stage or the blastocyst stage, on days 5-6 (2). On the other hand, better pregnancy outcomes could be attained by defining another standard for the simultaneous transfer of more than one embryo, resulting in multiple births, along with consequences such as abortion or premature birth (3).
Evidence suggests that if the blastocyst is transferred to the uterus instead of the embryo in the cleavage stage, as developmental stages are fully monitored in the laboratory, the possibility of synchronizing the uterus to accept the embryo will further increase (3). On the other hand it is very similar to a natural phase and allows embryo selfselection after stimulation of the embryonic genome on day 3 (4). Current advances in cell culture media have conducted to a shift in the early embryo whereas transfer process from the cleavage stage to the blastocyst stage in IVF (3,4). A recent study has indicated that since the morphological grade of the embryo in the cleavage and blastocyst stages cannot show chromosomal abnormalities; therefore, embryos with chromosomal defects may also progress to the blastocyst stage (5). Despite good reports on the prognosis of blastocyst embryo transfer (BET) programs, one study has shown that prolonged embryo culture, which can precede to a number of embryos failure to blastocyst stage, in vitro culture beyond embryonic genomic activation caused epigenetic effects on the embryo, and also an increased rate of transfer cessation and risk of monozygotic twins (4).
Embryo transfer was conventionally performed 2 days following oocyte retrieval, while changes in cell culture media allow embryos to be kept for longer times Delayed transfer from the 2 nd to the 3 rd day allows more fetal growth in vitro and may positively influence pregnancy outcomes (6). However, more retrospective and prospective studies are required to determine whether 2, 3, or 5-day-old embryo transfers differ in pregnancy outcomes.
Due to the contradictions in various studies regarding the consequences of embryo transfer in the cleavage and blastocyst stages, the present study aimed to retrospectively assess the records of the cases referred for embryo transfer to Dezful Infertility Center within the past 7 yr and review the outcomes of fresh embryo transfer in different stages of development.

Study design and participants
In this cross-sectional study, the files

Ovarian stimulation and oocyte retrieval
Using the antagonist protocol, controlled ovarian stimulation (COS) was performed as per the conventional method (7

Outcome measures
The determined outcomes contained a chemical pregnancy rate, which was considered as a serum

Statistical analysis
Data analysis was performed in Social Sciences software version 25.0 (SPSS Inc., Chicago, IL, USA).
Data were presented as mean ± standard deviation, count, and percentage. Due to a large number of samples and 4 groups in the present study, a oneway ANOVA parametric analysis of variance was used to compare the means in the studied groups, and the Chi-square test was used to compare the relationship between qualitative variables grouped.
In order to check the normality of the quantitative data, the non-parametric Kolmogorov-Smirnov test analysis was performed, which showed normal distribution of data (p < 0.05).

Results
A total of 1246 participants fulfilling the criteria defined in materials and methods participated in this study and received embryo transfer.  Table I).

Discussion
Recent advances in cell culture have led to a shift in IVF from embryo transfer in the initial stages of cleavage to embryo transfer in the blastocyst stage.
In terms of blastocyst culture and its transfer theory, this approach could improve the synchrony between the fetus and uterine endometrium. Our findings among 1246 eligible cases indicated no significant correlation between pregnancy outcomes and the embryo's developmental stage in embryo transfer cycles.
Research in participants aged > 36 yr, observed that BET compared to the cleavage stage was more likely to increase the probability of achieving a live birth. Also, they concluded that one good quality blastocyst reduces the likelihood of multiple pregnancies in those aged < 36 yr (11). Contrary to the findings of the mentioned study, the age of embryo transfer did not have a significant correlation with pregnancy outcomes in the current research.
This discrepancy could be due to the differences in sample size, population age, and the ratio of the transferred embryos in the cleavage and blastocyst stages.
In a study to investigate the possible impact of COS on the perinatal outcomes in 784 fresh transfers and 382 freeze-thawed dual blastocyst transfers showed clinically significant differences concerning the peri-implantation and perinatal outcomes of fresh and freeze blastocyst transfer, due to better endometrial receptivity and placentation in freeze-thawed cycles (12). This discrepancy could be due to the selection of fresh embryo transfer in this study that can be an effect of COS on implantation rate and pregnancy outcomes.
In another retrospective study of 11 However, embryo transfer in the blastocyst stage is associated with a reduced likelihood of multiple pregnancies (2). In this regard, although the rate of twin pregnancies did not decrease in our study, it did not differ significantly compared to other groups.
The difference in the number and quality of embryos transferred as well as the choice of patients can probably be justified.

Limitations and advantages
One of the limits of our analysis includes the retrospective kind. We were incapable of adjusting for several confounders, such as smoking status, body mass index. Also, this study failed to assess the cumulative live birth rate as the most valuable key performance indicator of the treatment. Also, perinatal outcomes were not included in our analyses. Advantage of the study we were able to assess the various stages of cleavage fresh embryo transfer and blastocyst stage on pregnancy outcomes. Also, all processes from the choice of cases, indications, IVF procedure, ovum pick up, and embryo transfer were done by one gynecologist that confounder caused by doing, was deleted.

Conclusion
According to the results, the developmental stage of the fresh embryo (blastocyst or cleavage stage) for transfer had no positive or negative effect on fertility. It seems BET is not superior to cleavage embryo transfer in reproductive outcomes.
However, it is suggested that similar investigations can be conducted in this regard on larger sample sizes to assess the different stages of embryo transfer, pregnancy, and perinatal outcomes.